2-(2-ethoxyethoxy)ethyl acetate

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions. Lack of test material details and raw data on clinical chemistry.

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

lack of test material details and raw data on clinical chemistry.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(2-ethoxyethoxy)ethyl acetate

EC Number:

203-940-1

EC Name:

2-(2-ethoxyethoxy)ethyl acetate

Cas Number:

112-15-2

Molecular formula:

C8H16O4

IUPAC Name:

2-(2-ethoxyethoxy)ethyl acetate

Details on test material:

- Name of test material (as cited in study report): Ethanol, 2- (2-Ethoxyethoxy)-, acetate
- Analytical purity: no data
- Other: in the report it is also stated, that a combination aerosol was tested, whoever the combination is not named as the study report is sanitized.

Test animals

Species:

rat

Strain:

not specified

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Housing: The animals were housed continously in groups of 2-5 animals in Hazelton H-1000 exposure chambers.
- Diet: was removed during exposure
- Water: was removed during exposure

Administration / exposure

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Hazelton H-1000 exposure chamber
- Method of holding animals in test chamber: whole body exposure
- System of generating aerosols: Laskin nebulizers in each chamber were immersed in water bath at approximately 180 °F (82.2 °C) during each exposure to simulate the temperature under normal conditions of use. The output of the nebulizer went through a glass impactor to remove the larger particles before being diluted by the main air stream entering the exposure chamber.
- Temperature, humidity, pressure in air chamber: recorded every 30 min during exposure; 24.6 °C (mean), 66.5% (mean)
- Air change rate: 12
- Method of particle size determination: at least once daily with a cascade impactor.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Nominal concentration was calculated daily as the loss of weight of test material from the generator divided by the total air flow through the chamber. In addition, a limited number of filters were analyzed using FTIR to evaluate shifts in composition over time.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 h/day, 5 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0.05 mg/L air (nominal)

Remarks:

Target value

Doses / concentrations 2

Dose / conc.:

0.06 mg/L air (analytical)

Doses / concentrations 3

Dose / conc.:

0.15 mg/L air (nominal)

Remarks:

Target value

Doses / concentrations 4

Dose / conc.:

0.15 mg/L air (analytical)

Doses / concentrations 5

Dose / conc.:

0.5 mg/L air (nominal)

Remarks:

Target value

Doses / concentrations 6

Dose / conc.:

0.52 mg/L air (analytical)

Doses / concentrations 7

Dose / conc.:

50 mg/m³ air (nominal)

Remarks:

Target value

Doses / concentrations 8

Dose / conc.:

60 mg/m³ air (analytical)

Doses / concentrations 9

Dose / conc.:

150 mg/m³ air (nominal)

Remarks:

Target value

Doses / concentrations 10

Dose / conc.:

150 mg/m³ air (analytical)

Doses / concentrations 11

Dose / conc.:

500 mg/m³ air (nominal)

Remarks:

Target value

Doses / concentrations 12

Dose / conc.:

520 mg/m³ air (analytical)

No. of animals per sex per dose:

15 (core groups)
10 (additional ancilliary males in each group for specialized endpoints)

Control animals:

yes, concurrent no treatment

yes, sham-exposed

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily, during exposure each 30 min

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily before exposure

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: before necropsy
- Anaesthetic used for blood collection: Yes
- Animals fasted: Yes
- How many animals: 15/sex/group
- Parameters checked: White blood cell count (WBC), red blood cell count (RaC), haemoglobin (Hb), haematocrit (Het), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets and differential white blood cell count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: before necropsy
- Animals fasted: Yes
- How many animals:15/sex/group
- Parameters checked: Glucose, creatinine, urea nitrogen, uric acid, cholesterol, triglycerides, total bilirubin, total protein, albumin, globulin, A/G ratio, sorbitol dehydrogenase (SDH), alanine aminotransferase (ALT or SGPT), aspartate aminotransferase (AST or SGOT), alkaline phosphatase (ALP), Cl, Ca, Na, K, and P.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. Adrenals, brain, epididymides, heart, kidney, liver, ovaries, prostate, spleen, testes, thymus, and uterus. Wet and dry weights of the right middle lung lobe were also measured. Lung, trachea, larynx and portions of the kidney and liver were preserved in a glutaraldehyde-formalin fixative
HISTOPATHOLOGY: Yes. Sham-exposed controls and animals in the high-dose group: Adrenals, sternum, brain (3 sections), eye and optic nerve, heart, colon and duodenum, kidneys, liver, lung (left lobe), thigh muscle, sciatic nerve, spleen, ovaries, pancreas, submaxillary gland, stomach, testes, thymus, thyroid, tracheobronchial lymph nodes, nasal turbinates (4 sections), urinary bladder, gross lesions. In addition, the lungs and tracheobronchial lymph nodes of untreated controls and the two other, exposed groups were similarly processed.

Other examinations:

The ancillary animals were included to provide information on endpoints on the lung which could not be performed without using some of the lung tissue and thus preventing subsequent histopathology. They were anesthetized and used for pulmonary function tests (deflation pressure-volume curves and functional residual capacity).
After the pulmonary function tests, the rats were exsanguinated and the lung lobes were removed and weighed. The right middle and postcaval lobes were frozen and saved for analysis of hydroxyproline content as an index of collagen and development of fibrosis.

Statistics:

Numerical data were analyzed by use of SAS. a computerized statistical analysis system. Values for body weights, organ weights, lung volumes, and pulmonary hydroxyproline were compared by use of ANOVA (analysis of variance) and Duncan's multiple range comparison test. Data on haematology, serum chemistry, and sperm were compared with ANOVA and Tukey's studentized range test.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

530 mg/m³ (female): increased globulin and bilirubin relative to sham-exposed but not to untreated controls, not treatment related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

150 and 520 mg/m³: increased weight of right middle lung lobe, small red areas and plaques on the surface of the lungs.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

see Table 2 under "Any other information on results incl. tables".

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
No treatment-related abnormalities were noted.

BODY WEIGHT AND WEIGHT GAIN
Statistically greater weights in selected males at the beginning of exposures were observed but not considered to be treatment-related (see Table 1 and 2 under “Any other information on results incl. tables”).

HAEMATOLOGY
Total WBC tended to be higher with higher doses and were statistically different for males in the high-dose group relative to untreated controls (but not to sham-exposed controls). Among WBC counts, there tended to be a shift in the relative proportion of cell types, with an increased percent of segmented neutrophils and a decrease in lymphocytes (see Table 3 under "Any other information on results incl. tables").

CLINICAL CHEMISTRY
Globulin was significantly increased by 17% in females of the high-dose group relative to sham-exposed but not to untreated controls. Total bilirubin was increased in females of the high-dose group by 47% compared to sham-exposed controls, but was almost identical to untreated controls. These statistically significant differences were minor and did not appear to be related to exposures (additional details are in a not available report on serum chemistry). Strong statistical differences were found only in the females receiving the two higher concentrations (additional details are given in a not available report on haematology).

ORGAN WEIGHTS
Wet and dry weight of the right middle lung lobe increased in a concentration-related manner (see Table 4 under "Any other information on results incl. tables"), each was significantly increased in both sexes in the two higher dose groups. The relative increases in mean weights were similar in both sexes. Dry weight increased proportionately more than wet weight. No significant changes in organ weights were observed in any other organs.

GROSS PATHOLOGY
At necropsy, small red areas and plaques were observed on the surface of the lungs, primarily in exposed animals. All rats in the two higher dose groups had plaques. No other significant treatment-related gross lesions were observed (additional details are given in a not available pathology report).

HISTOPATHOLOGY:

LUNGS
The response of the lungs to the aerosol consisted of foamy macrophages in the alveoli and alveolar walls, very mild thickening of alveolar walls due to foamy macrophages and a mixed cell inflammatory infiltrate, and subtle Type 2 cell hyperplasia. The incidence is summarized in Table 5 under “Any other information on results incl. tables”. Other related changes in the lungs were observed, namely, subacute inflammation, hyperplasia of Type 2 epithelial cells in alveoli, and thickening of the alveolar walls. The inflammation consisted of an increase of lymphocytes and a few polymorphonuclear leukocytes in the interstitium and alveoli. The inflammation was focal and mild. It tended to increase in severity with dose and become more multifocal, although it still remained only a mild change. The nature and severity of the other responses changed with aerosol concentration. In the most severe case, 20 - 30% of the alveoli of rats exposed to 0.52 mg/L contained foamy macrophages. However, no aggregates were severe enough to fill any given alveolus and in the alveolar walls, there was no microscopic evidence of an increase in connective tissue fibers. The overall response appeared to be an increase of free cells, such as macrophages, to the increased load of panicles on the alveolar surface. The response was present with 0.06 mg/L, in mild form, and increased with concentration.

OTHER ORGANS
Enlarged tracheobronchial lymph nodes were noted at necropsy in 0, 0, 3, 10, and 28 animals of groups 1, 2, 3, 4 and 5, respectively. Microscopically, these lymph nodes contained foamy macrophages; the number of macrophages appeared to correlate well with the effects in the lung. Foamy macrophages were also seen in the anterior mediastinal and thymic lymph nodes. No treatment-related effects were seen in any of the other tissues examined, including the nasal passages.

OTHER FINDINGS
NUMBER AND MORPHOLOGY OF SPERM
The test substance did not adversely affect the number of testicular spermatids or epididymal sperm and did not alter the morphology of the sperm. Additional details are given in an unavailable report on evaluation of sperm.

PULMONARY FUNCTION AND HYDROXYPROLINE IN ANCILLARY ANIMALS
As seen with the right middle lobe alone in the core animals, wet weight increased in a concentration-related manner. The total weight in all 3 exposed groups was significantly greater than either control group and also different from each other. Compared to the two control groups, mean lung weights were increased by 12, 29, and 76% for groups 3, 4, and 5, respectively. Lung volumes were also measured in the ancillary animals. Residual volume (RV) is the volume remaining in the lungs at the end of a forced expiration. Functional residual capacity (FRC) is the volume remaining at the end of a normal exhalation. RV and FRC are measures of lung volume when the lungs are inflated to only a small amount of their total capacity. TLC is the total lung capacity, the volume at maximal expansion. Lung volumes from the present study are among the parameters summarized in Table 4 under "Any other information on results incl. tables". Body weight was equivalent among the groups, indicating that no significant differences showed have resulted from variation in body size. Most of the lung volumes were also unaffected by exposure except the 2 lowest volumes, functional residual capacity and residual volume with exposure to 0.52 mg/L. Each was significantly lower than the sham-exposed controls, but not significantly different from the untreated controls. The significance of these stastistical differences is marginal because 1) they exist only in relation to sham-exposed controls and 2) an outlier in the sham-exposed controls seems to have skewed the data for that group. Therefore, the author concluded that there was a trend toward a decrease in the lower lung volumes with exposure to 0.52 mg/L.
Quasistatic pressure-volume curves represent lung volumes at specific transpulmonary pressures as the lungs are slowly deflated from a maximal inflation to a maximal deflation. There were no treatment-related changes. Pulmonary compliance is derived from the pressure-volume curves as the change in lung volume over specified ranges of transpulmonary pressure. Compliance was unaffected by exposure. Specific compliance is the compliance over a given range of pressure divided by the absolute lung volume at the midpoint of that pressure range. The intent is to standardize compliance to lung volume. It also was unaffected by exposure.
Data on hydroxyproline in the postcaval lung lobe are summarized in Table 5. Pulmonary hydroxyproline, is a measure of the connective tissue fiber, collagen. The amount of hydroxyproline per lobe was significantly greater in rats exposed to 0.52 mg/L than the untreated controls; but there was not a significant increase above the sham-exposed group. The wet weight of the postcaval lobe, in contrast was significantly greater than both control groups for groups exposed to 0.15 or 0.52 mg/L. The wet weight in the high-dose group tended to increase more than the hydroxyproline content, leading to a statistically insignificant tendency for reduced µg OHPro/mg lung.

Effect levels

1

Key result

Dose descriptor:

NOAEC

Effect level:

520 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

2

Dose descriptor:

NOEC

Effect level:

150 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. Mean body weights of male rats.

Mean body weights [g] Week	Group 1 Room control 0 [mg/L]	Group 2 Sham control 0 [mg/L]	Group 3 Low dose 0.06 [mg/L]	Group 4 Mid dose 0.15 [mg/L]	Group 5 High dose 0.52 [mg/L]
1	433.0	440.0	453.0	452.0	455.0
2	460.0	465.0	476.0	469.0	478.0
3	476.0	490.0	503.0	494.0	502.0
4	498.0	504.0	525.0	519.0	513.0
5	518.0	520.0	539.0	534.0	524.0
6	533.0	534.0	555.0	547.0	540.0
7	549.0	550.0	571.0	565.0	550.0
8	559.0	559.0	582.0	574.0	563.0
9	577.0	571.0	588.0	582.0	576.0
10	584.0	582.0	597.0	586.0	589.0
11	590.0	593.0	606.0	598.0	597.0
12	593.0	601.0	617.0	608.0	605.0
13	606.0	612.0	627.0	624.0	616.0
14	602.0	601.0	611.0	611.0	603.0
Mean	541.3	544.4	560.7	554.5	550.8

*: P<0.05 from Group 1

Table 2. Mean body weights of female rats.

Mean body weights [g] Week	Group 1 Room control 0 [mg/L]	Group 2 Sham control 0 [mg/L]	Group 3 Low dose 0.06 [mg/L]	Group 4 Mid dose 0.15 [mg/L]	Group 5 High dose 0.52 [mg/L]
1	263.0	266.0	267.0	278.0	273.0
2	274.0	272.0	280.0	285.0	277.0
3	284.0	285.0	291.0	292.0	288.0
4	289.0	290.0	295.0	307.0	300.0
5	300.0	294.0	304.0	310.0	303.0
6	305.0	301.0	308.0	312.0	307.0
7	310.0	307.0	314.0	317.0	312.0
8	316.0	310.0	318.0	320.0	319.0
9	325.0	317.0	320.0	325.0	323.0
10	325.0	321.0	328.0	330.0	325.0
11	335.0	326.0	332.0	335.0	330.0
12	340.0	328.0	335.0	336.0	337.0
13	337.0	334.0	340.0	342.0	340.0
14	327.0	322.0	328.0	327.0	328.0
Mean	309.3	305.2	311.4	315.4	311.6

Table 3. Segmented neutrophiles and lymphocytes as percent of total white blood cells and total number of white blood cells (thousands/µl).Values are mean ± SD.

	Group 1 Room control 0 [mg/L]	Group 2 Sham control 0 [mg/L]	Group 3 Low dose 0.06 [mg/L]	Group 4 Mid dose 0.15 [mg/L]	Group 5 High dose 0.52 [mg/L]
Males
WBC	13.6 ± 2.5	14.9 ± 2.2	14.5 ± 2.9	16.8 ± 3.1	17.5 ± 3.9a
Segmented neutrophiles	14 ± 6	17 ± 6	17 ± 11	15 ± 6	22 ± 6a
lymphocytes	84 ± 6	81 ± 5	82 ± 12	84 ± 6	77 ± 6
Females
WBC	10.9 ± 2.7	9.3 ± 2.8	10.3 ± 2.0	11.0 ± 2.2	12.1 ± 2.8
Segmented neutrophiles	13 ± 4	12 ± 6	15 ± 7	22 ± 6bd	25 ± 7bd
lymphocytes	86 ± 5	87 ± 6	83 ± 7	77 ± 6bd	74 ± 7bd

Significantly different from Group 1: a= p<0.05; b =p<0.01, different from Group 2: d=p<0.01

Table 4. Wet and dry weights (mg) and dry/wet ratio (%) of right middle lung lobe. Values are mean ± SD.

Weight	Group 1 Room control 0 [mg/L]	Group 2 Sham control 0 [mg/L]	Group 3 Low dose 0.06 [mg/L]	Group 4 Mid dose 0.15 [mg/L]	Group 5 High dose 0.52 [mg/L]
Males
Wet	185 ± 27	192 ± 32	197 ± 21	226 ± 26a	310 ± 40a
Dry	35 ± 4	36 ± 4	40 ± 4b	49 ± 5a	82 ± 13a
Dry/Wet	19.0 ± 1.4	19.1 ± 2.1	20.4 v 1.4	21.8 ± 1.7a	26.4 ± 1.7a
Females
Wet	132 ± 25	130 ± 19	140 ± 14	164 ± 16a	220 ± 18a
Dry	25 ± 4	26 ± 6	28 ± 4	35 ± 4a	59 ± 6a
Dry/Wet	19.2 ± 1.7	20.3 ± 5.3	19.8 ± 2.0	21.7 ± 2.3	26.8 ± 2.2a

a) Significantly different from both control groups.

b) Significantly different only from untreated controls.

Table 5. Incidence of microscopic changes in left lung lobe, 15 animals examined/sex.

	Group 1 Room control 0 [mg/L]	Group 2 Sham control 0 [mg/L]	Group 3 Low dose 0.06 [mg/L]	Group 4 Mid dose 0.15 [mg/L]	Group 5 High dose 0.52 [mg/L]
	Males
1-2 FAM	0	0	15	15	15
Occasional AGGs	0	0	9
Few AGGs	0	0		15
Many AGGs	0	0			15
FAM in alveolar walls	0	0	15	15	15
Type 2 cell hyperplasia	0	0	9	15	15
Occasional thick alveolar walls	0	0	7	15
Some thick alveolar walls	0	0			15
Subacute inflammation	2	2	0	2	7
	Females
1-2 FAM	0	0	15	15	15
Occasional AGGs	0	0	5
Few AGGs	0	0		14
Many AGGs	0	0			15
FAM in alveolar walls	0	0	15	15	15
Type 2 cell hyperplasia	0	0	5	15	15
Occasional thick alveolar walls	0	0	5	15
Some thick alveolar walls	0	0			15
Subacute inflammation	0	5	0	3	11

FAM = foamy alveolar macrophages

AGG = Aggregates of 4 or more foamy alveolar macrophages

Occasional AGGs = 2-3 FAM in < 10% of alveoli

Few AGGs = 2-3 FAM in 10-20% of alveoli

Many AGGs = More FAM7alveolus, > 20 % of alveoli with FAM, aggregates not large enough to fill any alveolus

Occasional thick alveolar walls = Mild thickening in a few alveolar walls

Some Thick alveolar walls = Increased thickening, but still mild; no increase in visible connective tissue in walls

Table 6. Hydroxyproline in postcaval lung lobe and weight of the lobe.Values are mean ± SD.

	Group 1 Room control 0 [mg/L]	Group 2 Sham control 0 [mg/L]	Group 3 Low dose 0.06 [mg/L]	Group 4 Mid dose 0.15 [mg/L]	Group 5 High dose 0.52 [mg/L]
µg OHPro	347 ± 102	412 ± 104	456 ± 161	440 ± 137	478 ± 83a
Wet weight (mg)	163 ± 18	179 ± 21	191 ± 20a	214 ± 25b	291 ± 24b
µg OHPro/mg PCL	2.1 ± 0.4	2.3 ± 0.5	2.3 ± 0.7	2.1 ± 0.6	1.7 ± 0.3

a = Significantly different from group 1

b = Significantly different from group 1 and 2

Applicant's summary and conclusion

Conclusions:

Exposure concentrations as high as 520 mg/m³ resulted in mild histologic changes limited to the lung. The changes were expected results of the particle load resulting from the high aerosol concentrations and represented a nonspecific response to a relatively innocuous aerosol.